Electrocatalytic Hydrocarbon Hydroxylation by Ethylbenzene

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Electrocatalytic Hydrocarbon Hydroxylation by Ethylbenzene Dehydrogenase from Aromatoleum Aromaticum Palraj Kalimuthu, Johann Heider, Daniel Knack, and Paul Vincent Bernhardt J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/jp512562k • Publication Date (Web): 30 Jan 2015 Downloaded from http://pubs.acs.org on February 2, 2015

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The Journal of Physical Chemistry

Electrocatalytic Hydrocarbon Hydroxylation by Ethylbenzene Dehydrogenase from Aromatoleum aromaticum Palraj Kalimuthu,1 Johann Heider2, Daniel Knack2 and Paul V. Bernhardt1,* 1

School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, 4072, Australia

2

Laboratory for Microbial Biochemistry and Synmikro Center for Synthetic Microbiology, Philipps University Marburg, 35043 Marburg, Germany E-mail: [email protected]

Abstract We report the electrocatalytic activity of ethylbenzene dehydrogenase (EBDH) from the βproteobacterium Aromatoleum

aromaticum. EBDH is

a complex 155

kDa heterotrimeric

molybdenum/iron-sulfur/heme protein which catalyses the enantioselective hydroxylation of nonactivated ethylbenzene to (S)-1-phenylethanol without molecular oxygen as co-substrate. Furthermore, it oxidizes a wide range of other alkyl-substituted aromatic and heterocyclic compounds to their secondary alcohols. Hydroxymethylferrocenium (FM) is used as an artificial electron acceptor for EBDH in an electrochemically driven catalytic system. Electrocatalytic activity of EBDH is demonstrated with both its native substrate ethylbenzene and the related substrate p-ethylphenol. The catalytic system has been modelled by electrochemical simulation across a range of sweep rates and concentrations of each substrate, which provides new insights into the kinetics of the EBDH catalytic mechanism.

1

ACS Paragon Plus Environment


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Introduction Molybdenum is an essential element for most life forms as it is required by enzymes catalyzing redox reactions relevant to carbon, sulfur and nitrogen metabolism.1-3 Apart from the multinuclear enzyme nitrogenase, Mo is bound to the bidentate molybdopterin ligand cofactor (Scheme 1) where it is involved in coupled two-electron, O-atom transfer reactions of a range of substrates.3 The relevant metal centred oxidation state changes are either O=MoVI → MoIV (substrate oxidation) or MoIV → O=MoVI (substrate reduction), with the oxido ligand often, but not always, exchanged with the substrate. On the basis of their sequences and of structural differences at the active site Mo enzymes are classified into three major families such as xanthine oxidase family, sulfite oxidase family and DMSO reductase family.4 Dependent on the enzyme family, either one or two molybdopterins may be bound per Mo, and the dithiolene chelating group(s) is (are) essential in tuning the redox properties of the respective active sites. One of the more recently discovered Mo-enzymes is ethylbenzene dehydrogenase (EBDH) isolated from the β-proteobacterium Aromatoleum aromaticum. which is involved in the direct anaerobic oxidation of non-activated ethylbenzene to 1-(S)-phenylethanol.5-8 Furthermore, EBDH catalytically converts a broad spectrum of aromatic and heterocyclic compounds with ethyl or propyl substituents to their secondary alcohols with high enantioselectivity and thus it is potentially useful in the generation of precursors to fine chemicals.9-11 It is the first known enzyme capable of the direct anaerobic oxidation of non-activated hydrocarbons.8

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FS0 FS2 Mo FS1

FS4 FS3

heme

Figure 1. Crystal structure of EBDH (PDB code 2IVF)12 showing the seven redox active cofactors.

EBDH consists of three subunits of molecular masses of 96, 43 and 23 kDa. The largest subunit contains the Mo active site ligated by two molybdopterin guanine dinucleotides and an Fe-S cluster (FS0, Fig. 1). The middle subunit bears three [4Fe-4S] clusters (FS1-FS3) and a single [3Fe-4S] cluster (FS4) while the smallest subunit contains a heme b.12 EBDH belongs to the DMSO reductase family and exhibits high sequence similarities with other enzymes such as selenate reductase from Thauera selenatis, dimethylsulfide dehydrogenase from Rhodovulum sulfidophilum and chlorate reductase from Dechloromonas sp.4, 13-15 These enzymes belong to a group collectively known as the complex iron sulfur molybdoenzymes (CISMs).16 In this case the two electrons resulting from ethylbenzene oxidation at the Mo active site flow (one at a time) from left to right as shown in Figure 1 via the five Fe-S clusters to the heme.

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The crystal structure of EBDH also revealed a relatively large active site cavity which permitted entry of several aromatic substrates containing a variety of substituents in different positions. Unsaturated substituents and more bulky bicyclic compounds such as p-ethylbiphenyl and 2ethylnaphthalene are also active substrates. 9, 12, 17 Asp223 CH3 CH3 ethylbenzene H

H

H

H

S S

N

O

MoVI

O

H

O Asp223

S

S

S CH3S

N

H

Asp223 S S

MoVI

O N

H

O

MoV

O

S

HO

H

His192

O

S

N

O

N His192

S

S Asp223

N His192

S +

2H , 2e

-

S Asp223 S S H 2O N N His192

MoIV

O

MoIV

CH3HO H

O

H

O

O

S

S

N H 2O

S

N His192

S

H

CH3 H

OH

S-1-phenylethanol

Scheme 1. Proposed catalytic mechanism18, 19 of ethylbenzene hydroxylation by EBDH.

The catalytic mechanism of Mo containing enzymes are generally associated with oxido ligand transfer reactions in which an O-atom is exchanged directly between the Mo active site and the substrate coupled with 2-electron transfer with the metal.4, 20-23 In contrast, the proposed EBDH catalytic mechanism is more complicated.10, 18 Based on quantum chemical modeling and embedding this model 4


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into the EBDH structure (QM/MM model), the mechanism shown in Scheme 1 was proposed.10, 17, 19, 24, 25 The Mo active site can be reached through the 25 Å long tunnel shaped pocket lined by mostly nonpolar residues.12 On the basis of crystallographic evidence Asp223 is coordinated to the Mo ion and His192 is a potential H-bond donor to the predicted oxido ligand in oxidized EBDH.10, 18 The proposed mechanism involves H-abstraction generating an intermediate comprising an ethylbenzyl radical and a hydroxido-coordinated MoV ion followed by a second single electron transfer from the radical to MoV to generate a second intermediate involving MoIV and a ethylbenzyl secondary carbocation. The OH- ligand rebounds by combining with the adjacent carbocation to form the product 1-(S)-phenylethanol. The enantioselectivity of this transformation is typically very high across a number of related substrates.9, 10 The redox potentials of EBDH have been determined26 and they follow the same trend reported for the highly homologous DMS dehydrogenase (R. sulfidophilum).27 The highest potential cofactor is the heme (Eo = +255 mV vs NHE)26 to which electrons are ultimately drawn after substrate oxidation, although several of the intermediate redox transitions via the Fe-S clusters are endergonic. The heme is clearly the site of electron egress from EBDH and it is this site that interacts with electron acceptors both in vivo and in synthetic systems. The ferrocenium ion has been found to be an effective synthetic electron partner for EBDH in solution assays.6, 9 Coupling of the EBDH catalytic reaction to an electrochemically regenerated electron acceptor has recently been reported to allow bulk scale hydroxylation.11 In the present study, we report the mediated electrocatalytic voltammetry of EBDH with two different substrates, namely ethylbenzene and p-ethylphenol, in the presence of the synthetic redox mediator hydroxymethylferrocene (ferrocenemethanol, FMox/red, +420 mV vs NHE, Scheme 2) employing a Au working electrode modified with the heterocyclic thiol 5-(4’-pyridinyl)-1,3,4-oxadiazole-2-thiol (Hpyt). Digital simulation was employed to explore the kinetics of the coupled homogeneous chemical reactions at a variety of sweep

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rates as well as different substrates concentrations and it provides new insights to the EBDH catalytic mechanism.

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Experimental Section Materials. EBDH was purified from cells of strain EbN1 (Aromatoleum aromaticum) grown on ethylbenzene as previously described.5 Ferrocene methanol (FM) was synthesized by based on the previously reported procedure28 in which commercially available ferrocene carbaldehyde is reduced by NaBH4 in methanol, washed with water extracted into chloroform and evaporated to dryness. Ethylbenzene, p-ethylphenol and toluene were purchased from Aldrich and were used as received. All other reagents used were of analytical grade purity and used without any further purification. The stock solutions of ethylbenzene, p-ethylphenol and toluene were prepared in tert-butyl alcohol. All other solutions were prepared in purified water (Millipore, resistivity 18.2 MΩ.cm). Phosphate buffer solution was prepared using 50 mM Na2HPO4 and NaH2PO4. A mixture of buffers (10 mM MES, 10 mM Bis-Tris, 10 mM Tris and 10 mM CHES) was used for pH dependent experiments across the range 5.5 < pH < 9.5 and the desired pH was obtained by addition of dilute acetic acid or NaOH.

Electrochemical Measurements and Electrode Cleaning. Cyclic voltammetry (CV) was carried out with a BAS 100B/W electrochemical workstation using a three-electrode system consisting of a Au working electrode, a platinum wire counter electrode, and a Ag/AgCl reference electrode. Potentials are cited versus normal hydrogen electrode (NHE). The gold working electrode was first cleaned according to a published protocol29 then modified with 5-(4’-pyridinyl)-1,3,4-oxadiazole-2-thiol as described.30 A platinum wire counter electrode and Ag/AgCl reference electrode were used (+196 mV vs NHE). Unless otherwise stated, electrochemical solutions were purged with argon for at least 30 min prior to the series of experiments and all experiments were performed under a blanket of argon gas. The electro-active surface area of the Au electrode (A = 0.055 cm2) was determined by CV of 1 mM ferrocene methanol (FM) in 0.1 M KCl solution at different sweep rates using the Randles-Sevcik equation (eq. 1).31 The diffusion coefficient (Do) of FM is 6.7 × 10-6 cm2 s-1,32 ip is the measured current 7



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maximum, the number of electrons n=1, concentration of analyte Co = 10-6 mol cm-3, and ν is the sweep rate (V s-1).

ip = (2.69 × 105)n3/2ADo1/2Coν1/2

(1)

The variation of the observed limiting catalytic current (ilim) as a function of substrate concentration (Csub) followed Michaelis-Menten kinetics and the data were fit to the eq. (2).33 𝑖max [Sub] (2) 𝐾M,app + [Sub] where imax is the saturation limiting current and KM,app is the apparent Michaelis constant. 𝑖lim =

The pH dependence of the catalytic current was modeled by eq. (3) which is applicable for an enzyme that is deactivated by either deprotonation (pKa1) or protonation (pKa2).34 𝑖lim (pH) =

1+

𝑖opt (pH−p𝐾 a1 ) + 10

10(p𝐾a2 −pH)

(3)

Enzyme Electrode Preparation. A 5 µL aliquot of 21 µM EBDH in Tris-HCl buffer solution (pH 8) was dropped on a freshly prepared, inverted Au/Hpyt electrode and this was allowed approximately 30 min to dry to a thin film at 4 oC in the refrigerator. To prevent protein loss the electrode surface was carefully covered with a perm-selective dialysis membrane presoaked in water. The dialysis membrane was pressed onto the electrode Teflon cap and fastened to the electrode with a rubber O-ring to prevent leakage of the internal membrane solution. The resulting modified electrode was stored at 4 oC in 50 mM phosphate buffer solution (pH 7.0) when not in use.

Electrochemical Simulation. The DigiSim (version 3.03b) program was employed35 to simulate the experimental cyclic voltammograms of FM obtained at different sweep rates and different substrate concentrations. The experimental parameters restrained in each case were the working electrode surface area (0.055 cm2) and the double-layer capacitance (2 µF). Semi-infinite diffusion was assumed and all pre-equilibration reactions were disabled. The diffusion coefficients for EBDH (all forms) and substrates (ethylbenzene and p-ethylphenol) were 9×10-8 and 7×10-7 cm2 s-1 respectively. The apparent 8


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diffusion coefficient used in the simulation for FM (5×10-7 cm2 s-1) is one order of magnitude lower compared to the reported experimental value used in equation 1 (6.7×10-6 cm2 s-1).32 This value effectively models the attenuated mass transport of FM from the bulk, across the membrane to the diffusion layer as described previously.36 The heterogeneous rate constant was 0.05 cm s-1 determined from simulating the sweep rate dependence of the anodic peak to cathodic peak separation (in the absence of EBDH). The apparent redox potential of FM+/0 was determined from a control experiment without substrate present. All of the above mentioned parameters were then held constant during simulations of different sweep rate and substrate concentration dependent catalytic voltammograms and only the homogenous rate constants (k1-k4; k-1-k-4) in Scheme 2 were allowed to vary.

1-phenylethanol N N

FMox

k-4 k3 k-3 EBDHred

e

k4

-

EBDHred:1-phenylethanol

N

O N N

S

N

O N N

S

N

O N N

S

N

O N N

S

N

O N N

S

N

O N N

S

N

O

S

FMred

k2 k-2

EBDHint FMox

k'-4 -

EBDHox:Ethylbenzene

e

EBDHox

k1

Ethylbenzene

k-

1

k'4

FMred

Au/pyt electrode Dialysis membrane

Scheme 2. Mediated Electrochemically Driven Catalysis of EBDH with ethylbenzene.

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Results and Discussion Mechanism for Mediated Electrocatalysis of EBDH. A simplified double substrate (pingpong) mechanism was used to model the reaction (Scheme 2). EBDH is trapped beneath the dialysis membrane in proximity to the electrode surface and reacts with both substrate and oxidized form of the mediator (ferrocenium, FMox). The substrate (ethylbenzene or p-ethylphenol) and mediator (FMred) in the bulk solution are under diffusion control but must cross the membrane to access the enzyme or electrode. FM is a single electron transfer mediator so two consecutive outer sphere redox reactions are required to fully restore EBDH to its active (oxidized) form. We have assumed that the EBDH-substrate reaction follows Michaelis-Menten kinetics and it is associated with the substrate binding (k1/k-1), turnover (k2/k-2), product release (k3/k-3) and reactivation by FMox (two consecutive, one-electron, outer sphere redox reactions k4/k-4 and k’4/k’-4). The rate constants as k1/k-1, k2/k-2 and k3/k-3 are mediator independent whereas k4/k-4 and k’4/k’-4 are substrate independent. The EBDH-FM reaction proceeds by an outer-sphere electron transfer mechanism and no pre-equilibrium between enzyme and mediator is considered. This is a simpler case than normally associated with a true ping-pong mechanism where non-covalent interactions would drive the formation of an enzyme-electron partner complex. FMox, FMred, EBDHox and EBDHred are the oxidized and reduced forms of mediator and enzyme, respectively.

Catalytic Voltammetry. An example of the mediated catalytic voltammetry of EBDH with pethylphenol is illustrated in Figure 2. It was reported that p-ethylphenol is a better substrate for EBDH than ethylbenzene due to the hydroxyl substituent in the para position, which may H-bond with Asp485.37 Although EBDH has seven electro-active cofactors (Mo, heme b and five iron-sulfur clusters, Figure 1)12 no ‘non-turnover’ redox responses were observed from these cofactors at the Au/Hpyt electrode (data not shown). Most of these cofactors are deeply buried inside the protein structure (Figure 1) and not accessible to the electrode surface.12 Alternatively the small molecular weight redox 10


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mediator FM is capable of shuttling electrons between EBDH and the electrode where its heterogeneous electron kinetics are facile.

0.2

b

a

0.1

I / µΑ

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0

–0.1 200

300

400

500

E / mV vs. NHE Figure 2. CVs obtained for 50 μM FM at Au/Hpyt/EBDH electrode in (a) absence and (b) presence of 200 µM p-ethylphenol in 50 mM phosphate buffer (pH 7) at a sweep rate of 5 mV s−1.

At the Au/Hpyt/EBDH electrode, in the absence of substrate, a reversible FMox/red redox wave at +420 mV is observed with a peak to peak separation of ca. 50 mV (Fig. 2a) in phosphate buffer (50 mM, pH 7) which is characteristic of a single electron transfer redox process.32 This current is governed by the diffusion of both oxidized and reduced forms of FM between the electrode and bulk solution via the membrane. Upon introduction of p-ethylphenol (400 µM) to the electrochemical cell a complicated amplified anodic wave emerges (Fig. 2b). This is indicative of a catalytic homogeneous reaction coupled to heterogeneous electron transfer (EC′ mechanism).31 Here p-ethylphenol is oxidized to p-(1hydroxyethyl)phenol37 yielding a reduced form of the enzyme EBDHred which is oxidized again by electrogenerated FMox at the electrode surface thus leading to an amplified anodic current through 11



mediator recycling. The catalytic waveform is dependent upon the diffusion of FMred to the Au/Hpyt electrode, reaction between p-ethylphenol and EBDHox, and the reaction between FMox and EBDHred. In a control experiment, we found that the response of 50 µM FM at the Au/Hpyt electrode, in the absence of EBDH, was unaltered by addition of 400 µM p-ethylphenol (Supporting Figure S1). This illustrates that p-ethylphenol is electro-inactive in the experimental potential window of +250 to +600 mV vs NHE and also it does not undergo any chemical reaction with FM in either its oxidised or reduced form. 0.2

0.2

B

A e

e 0.1

I / µΑ

a

0.1

I / µΑ

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a

0

0

–0.1 200

300

400

–0.1 200

500

300

400

500

E / mV vs. NHE

E / mV vs. NHE

Figure 3. CVs obtained for varying concentrations of (A) ethylbenzene and (B) p-ethylphenol in the presence of 50 µM FM at Au/Hpyt/EBDH electrode (a) 0, (b) 25, (c) 50, (d) 100 and (e) 200 µM substrate in 50 mM phosphate buffer (pH 7) at a sweep rate of 5 mV s-1.

Enzyme-Substrate Reaction. The EBDH - substrate reaction was examined by varying the concentrations of its natural substrate ethylbenzene (Figure 3A) and also of p-ethylphenol (Figure 3B) in the presence of 50 µM FM at the Au/Hpyt/EBDH electrode. At a concentration of 25 µM ethylbenzene, a catalytic pre-wave emerges at ca. +300 mV in the presence of 50 µM FM at Au/Hpyt/EBDH electrode (Figure 3A, curve b). The pre-wave grows steadily in magnitude relative to the higher potential 12


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(uncoupled) FMox/red wave as the substrate concentration rises (50 to 400 µM). The pre-wave is associated with the coupled catalytic EBDH-ethylbenzene reaction but depletion of ethylbenzene in the vicinity of the electrode (under the membrane) results in the CV reverting to that of the uncoupled reversible FMox/red response at ca. +450 mV when the supply of substrate is exhausted. Similar phenomena were reported by our group recently with the mediated voltammetry of other molybdoenzymes on different electrode surfaces.20, 38 It is apparent that the shape of the transient wave becomes more sigmoidal at higher ethylbenzene concentrations (400 µM) and the cathodic wave diminishes (Figure 3A, curve e). However, even at the highest substrate concentrations there is still a slight shoulder due to reduction of some persistent FMox in the diffusion layer. The kinetics of this reaction are considered in greater detail later. We also investigated the catalytic activity of Au/Hpyt/EBDH electrode towards p-ethylphenol (Figure 3B) with identical conditions as indicated in Figure 3A. At low p-ethylphenol concentrations a prewave emerges at ca. +300 mV which grows in magnitude with substrate concentration. The apparent Michaelis constant (KM,app) was calculated from the dependence of the high potential catalytic current from substrate concentration at pH 7 using equation 2. The anodic currents at +350 vs NHE were chosen for ethylbenzene and p-ethylphenol, respectively, which increase linearly up to ca. 100 μM substrate and then approach an asymptote at around 400 μM for both ethylbenzene and p-ethylphenol (Supporting Figure S2). The calculated KM,app values were found to be 81 and 102 µM for ethylbenzene and p-ethylphenol, respectively. The KM,app value obtained here for p-ethylphenol is about 2.5-fold higher than that found from spectrophotometry (40 µM) using ferrocenium tetrafluoroborate as an electron acceptor.9, 17 However, the KM,app value obtained for ethylbenzene (81 µM) is more than 100-fold higher than the published value from a solution assay using stopped-flow spectrophotometry

13



(0.45 µM).17 The variation is most likely attributable to mass transport limitations of the substrate and mediator crossing the membrane as observed previously.36

0.18 0.16 0.14 0.12

I / µA

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0.10 0.08 0.06 0.04 0.02 0.00

-0.02

4

5

6

7

pH

8

9

10

11

Figure 4. pH dependence of the maximum oxidation current in the presence of 400 μM ethylbenzene at the Au/Hpyt/EBDH electrode at a sweep rate of 5 mV s−1. The solid curve is obtained from a fit to the experimental points using Eq. (3).

pH Profile. The electrocatalytic activity of Au/Hpyt/EBDH electrode towards the oxidation of ethylbenzene was investigated within the range of pH 5.5 to pH 9.5 in 40 mM mixed buffer solution. Figure 4 illustrates the activity/pH profile. The catalytic current exhibits a pH optimum of 8 which is similar to that obtained from the solution assay using ferrocenium tetrafluoroborate as electron acceptor (pH optimum 7.5).18 The bell shaped catalytic profile was modeled with equation 3 for a system that is deactivated by either protonation (pKa1 = 5.9) or deprotonation (pKa2 = 9.8). Furthermore, the pH dependence was reversible and independent of the direction of titration. Catalytic activity was fully restored when the pH was returned to its optimal value. 14


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a 0.2

I / µΑ

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0.1

b

0

300

400

500

600

E / mV vs. NHE Figure 5. CVs obtained for 400 µM ethylbenzene and 50 µM FM in the (a) absence and (b) presence of 5 mM toluene at Au/Hpyt/EBDH electrode in 50 mM phosphate buffer (pH 7) at a sweep rate of 5 mV s-1.

Catalytic Inhibition. It has been reported that several compounds including toluene, indole, styrene and anisole act as effective competitive inhibitors of EBDH as they bind strongly to active site but do not undergo hydroxylation thus blocking access by other substrates.9 As shown in Figure 5a the initial catalytic activity of the Au/Hpyt/EBDH electrode in the presence of FM and ethylbenzene (400 µM) is greatly suppressed upon addition of 5 mM toluene (Figure 5b) leaving only the transient FMox/red response now uncoupled from the inhibited EBDH/ethylbenzene reaction.

Simulation of the Catalytic Voltammetry The ultimate goal of electrochemical simulation is to deduce the kinetic constants defined in Scheme 2 that reproduce all voltammetry performed at different sweep rates and concentrations of each substrate. The coupled homogeneous chemical reactions are mostly bimolecular so the concentrations of each reactant will have an effect on the observed reaction rates and CV. The relevant values derived from the simulations are given in Table 1.

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A

0.2 µΑ

B

0.2 µΑ

–1 2 mV s

–1 2 mV s

–1 5 mV s

–1 5 mV s

–1 10 mV s

–1 10 mV s

–1 20 mV s

–1 20 mV s

–1 50 mV s

–1 50 mV s

200

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300

400

500

200

300

400

500

E / mV vs. NHE

E / mV vs. NHE

Figure 6. Experimental (solid lines) and simulated (dotted lines) CVs for (A) 10 µM FM and 200 µM ethylbenzene and (B) 50 µM FM and 50 µM ethylbenzene at the Au/Hpyt/EBDH electrode in 50 mM phosphate buffer solution (pH 7) at different sweep rates.

Table 1. Kinetic Parameters Derived from Electrochemical Simulation (published values in brackets). Ethylbenzene p-ethylphenol -1 -1

k1 (M s ) k-1 (s-1) k2 (s-1) k-2 (s-1) k3 (s-1) k-3 (M-1 s-1) k4 (M-1 s-1) k-4 (M-1 s-1) k’4 (M-1 s-1) k’-4 (M-1 s-1)

1.0 × 105 100 2 (1) 4.2 × 10-3 0.77 1.5 × 10-3

5.0 × 105 200 2 (4.8) 4.2 × 10-3 1 2.1× 10-3 7.0 × 106 1.4 × 102 7.0 × 106 1.4 × 102

Sweep Rate and Mediator Concentration Dependence. The voltammetric sweep rate is a powerful variable as it defines the timescale of each experiment. At high sweep rates all homogeneous 16


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reactions are bypassed and only the reversible heterogeneous electron transfer reaction (the FMox/red couple) will be actuated. As the sweep rate is decreased, electro-generated FMox in the reaction layer close to the electrode surface has time to react with EBDHred (the product of substrate turnover) and the CV takes on a more complex waveform. Under these conditions FMred is regenerated by EBDHred (as well as the intermediate half reduced form EBDHint) then electrochemically reoxidized which leads to an amplification of the anodic current and concomitant loss of the cathodic wave altogether because FMox formed at the electrode surface is continually consumed by the enzyme. Under ideal conditions an electrochemical steady state results and a sigmoidal waveform is observed. In effect the electrochemical reaction becomes biased completely toward oxidation. Here an ideal steady state is never seen due to a combination of substrate and mediator mass transport limitations. The sweep rate dependent CVs of the Au/Hpyt/EBDH/FM system in the presence of 200 μM ethylbenzene and two different concentrations of FM (10 μM in Figure 6A and 50 μM in Figure 6B) are illustrated. At the higher bulk FM concentration (Figure 6B) a cathodic peak around 360 mV emerges with increasing sweep rate due to accumulation of FMox in the reaction layer. A combination of the faster sweep rate (greater flux of FM to the electrode) and the shorter timescale of the sweep (uncoupling the FMox:EBDHred reaction) results in this buildup of FMox at the electrode at high potential and the observation of a significant return cathodic peak current. In Figure 6A the effect of the lower FM concentration (10 μM) is clear. Firstly, no cathodic wave is seen even at the highest sweep rate because FMox is always the limiting reagent and is immediately consumed by EBDHred and EBDHint at this lower FM concentration (10 μM). Secondly only a single anodic peak is observed which corresponds to the prewave seen at higher FM concentrations. No reversible FMox/red response is observed at +420 mV as the concentration of FMred at the electrode surface is very low and the EBDH:FM reaction is tightly coupled. In this case the rising prewave is due to the catalytic ethylbenzene oxidation which then collapses due to depletion of ethylbenzene from the reaction layer 17



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(enzyme oxidation outrunning mass transport). At this point catalysis is halted and the prewave is replaced by the transient, reversible FMox/red couple. All other sweep rate dependent simulated CVs recorded at various ethylbenzene and pethylphenol concentrations are given in the Supporting information (Figures S3 to S14). Also of note is that the anodic wave appears to shift to higher potential as the sweep rate increases. This can only be understood by realising that the waveform is a convolution of two different processes; FMred diffusing from the bulk straight to the electrode plus FMred ‘rebounding’ from EBDHox back to the electrode for reoxidation after homogeneous electron transfer. The homogeneous outer sphere redox reactions between FMox and the one and two-electron reduced forms of EBDH (EBDHred k4 and EBDHint k4’ ) were the same. In this case the (ferrous) heme cofactor (see Figure 1) is the likely group that reacts with FMox with intramolecular electron transfer within EBDH being non-rate limiting. The heme bears the highest redox potential within EBDH26, 27 and is the thermodynamic sink for electrons generated by substrate oxidation. The FMox-EBDH reaction should be independent of substrate (see Scheme 2). Substrate concentration dependence. Figure 7 depicts the effect of increasing concentrations of ethylbenzene (Figure 7A) and p-ethylphenol (Figure 7B) in presence of 50 µM FM at Au/Hpyt/EBDH electrode. As shown in Figure 7A, at low ethylbenzene concentrations the reversible high potential (uncoupled) FMox/red wave is dominant along with a small prewave which collapses at higher potential due to the depletion of substrate as discussed above. It is apparent that the reversible uncoupled FMox/red wave is gradually subsumed by the prewave with increasing substrate concentrations as the FMox-EBDHred reaction grows in significance thus scavenging all FMox from the reaction layer before it can be reduced on the cathodic sweep. At the substrate concentrations shown in Figure 7 the most obvious feature is that the prewave appears to move to higher potential with increasing concentration

18


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but this is actually a result of the catalytic reaction being sustained further along the potential axis by the greater supply of substrate from the bulk.

A

B

0.1 µΑ

25 µΜ

25 µΜ

50 µΜ

50 µΜ

200

0.1 µΑ

100 µΜ

100 µΜ

200 µΜ

200 µΜ

300

400

200

500

E / mV vs. NHE

300

400

500

E / mV vs. NHE

Figure 7. Experimental (solid lines) and simulated (dotted lines) CVs for varying concentrations of (A) ethylbenzene and (B) p-ethylphenol in the presence of 50 µM FM at the Au/Hpyt/EBDH electrode in 50 mM phosphate buffer solution at a sweep rate of 5 mV s-1. The deviations in absolute current between simulation and experiment at high potentials (> +400 mV) are due to a small background contribution from water oxidation.

Analysis of Kinetics Parameters. The kinetics parameters were obtained from electrochemical simulation in Table 1 and compared with some of the values reported by conventional solution assays with ferrocenium tetrafluoroborate as the electron acceptor.9, 17 The k4/k-4 and k’4/k’−4 values (EBDH reaction with FM) must be independent of substrate, while the k1/k-1, k2/k-2 and k3/k-3 values (EBDH reaction with substrates/products) may differ for each substrate. The simultaneous determination of multiple variables is problematic in any simulation due to some parameters having no effect on the simulated CV in some cases. Most steps were practically irreversible so the reverse rate constants (k−2, k−3, k−4 and k’−4) are only estimates i.e. some of the values are merely upper bounds. 19



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There was very little variation in the substrate dependent parameters as might be expected from the similarity of the CVs (see Figure 7). The substrate binding rate (k1) of ethylbenzene was somewhat lower than p-ethylphenol. There are no comparable values in the literature for this system. Given the uncertainty introduced by mass transport limitations of the membrane,36 through which both substrates must pass, we cannot attribute the apparent differential ‘on-rates’ for each substrate to structural interactions with EBDH. The observed deviation of the KM,app values from those determined by solution assays can be rationalized if the values of k1 obtained from the simulations are indeed lower than their true values (by an order of magnitude). Nevertheless the values determined here for k1 are lower bounds. A five-fold faster turnover number (k2) of p-ethylphenol over ethylbenzene has been reported previously from solution assays.9 Molecular modelling has suggested that the hydroxyl group of the substrate may H-bond with Asp485,37 which may accelerate the hydroxylation reaction and lead to resonance stabilisation of the carbocation intermediate. The outer sphere electron transfer rate constants k4 and k’4 are well-defined because they the simulations are very sensitive to these parameters at both lower and higher concentration of substrates and mediator. The reactions are particularly fast compared with recent systems that we have investigated and demonstrate that FM is an ideal synthetic electron partner for EBDH.

Conclusions The mediated electrocatalytic voltammetry of EBDH in reactions with its native substrate ethylbenzene and also with p-ethylphenol was demonstrated using FM as an artificial electron acceptor for the first time. Both transient and sigmoidal voltammograms were obtained depending upon the substrate concentrations and sweep rates. Catalytic inhibition was demonstrated with the competitive inhibitor toluene. A bell shaped catalytic pH profile was obtained with an optimal value of pH 8 for EBDH in reaction with ethylbenzene. Moreover, digital simulation enabled to determine a single set of 20


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apparent rate and equilibrium constants that modelled the electrochemical system over all experimental conditions.

Acknowledgement We gratefully acknowledge financial support from the Australian Research Council (DP120101465 & DP150104672), the German research foundation and the Synmikro Centre Marburg.

Supporting Information Available. Control experiments are shown for the electrochemical activity of p-ethylphenol on a Au/Hpyt electrode. Michaelis-Menten plots are shown for increasing concentrations of the substrates ethylbenzene and p-ethylphenol. Comparisons of experimental and simulated voltammetry are shown at various sweep rates for both ethylbenzene and p-ethylphenol.

21



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References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

15. 16. 17.

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Table of Contents Graphic

1-(S)-phenylethanol

ethylbenzene

CH3

CH3 H

H

H

OH

Asp223 S S

O

MoVI

O

O

Asp223 H 2O

S S

S

H 2O

S -2H

+

2 FMox

2 FMred

electrode 2e

-

24


MoIV S

O S

O

Electrocatalytic Hydrocarbon Hydroxylation by Ethylbenzene

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